During electrophoresis of biomolecules such as protein and nucleic acids (DNA & RNA), the biomolecules separate into distinct bands. One of the methods for isolation of a single species of protein, nucleic acids, and the like is electrophoresis followed by electroelution of a single electrophoresis band. There are various devices currently available for electroelution but they all include one common step: the band for electroelution must first be cut away from the separating gel and then loaded for electroelution in an electroelution device. Electroelution devices are engineered distinctly separate from the devices used for analytical electrophoresis and the use of such an electroelution device requires additional preparation and additional steps such as, setting up the device, preparation of reagents, loading of samples, setting up the running condition, and a procedure for recovery of electroeluted samples. Electroelution has historically been a laborious method, and the devices used have been difficult to operate.
Analytical electrophoresis of nucleic acids is most commonly performed by submerged horizontal electrophoresis. As the name implies, a gel is submerged horizontally in an electrophoresis tank. Samples are applied in preformed wells in the gel, and when electrical current is applied across the gel, nucleic acid molecules migrate toward the positive terminal and separate into distinct size-dependent bands. In the prior art, recovery of the nucleic acid from any of the bands required excision of the band followed by either chemical extraction or electroelution in a separate electroelution device.
As disclosed in the parent, following electrophoretic separation of biomolecules such as protein, nucleic acids and the like into distinct bands, electroelution is performed by engaging a piece of the separating gel containing the band of biomolecules intended for electroelution in a tubular enclosure. Preferably, the tubular enclosure has a rectangular cross section and the enclosure interior is substantially the same shape and size as the piece of separating gel containing the band of biomolecules intended for electroelution. Having a tubular enclosure with an interior shape and size substantially the same as the piece of separating gel containing the band of biomolecules intended for electroelution forms a piece of separating gel which fits into the tubular enclosure like a plug. The gel piece forms a substantially liquid impervious seal with the enclosure and partitions the buffer in the tubular enclosure from the buffer between the piece of gel and the closure means. After electroelution, the buffer in the tubular enclosure can be removed without disturbing the electroeluted biomolecules in the closure means. Such a tubular enclosure eliminates the need for a septum in the tubular enclosure for separating the gel pieces in the tubular enclosure from the buffer in the closure means. The piece of separating gel itself acts as a septum. Furthermore, the tubular enclosure is minimized to the size and volume equal to the piece of separating gel. It is also possible to have a tubular enclosure in which a blank piece of gel (i.e., a gel piece without any molecules for electroelution) is engaged. In this case, the blank piece of gel acts as a plug, and the pieces of gel having molecules for electroelution are loaded behind the blank piece of gel.
The tubular enclosure is provided with a closure means for preventing the passage of biomolecules (such as nucleic acid and the like) out of the tubular enclosure without hindering the electrophoresis electrical field. The closure means may be either an integral part of the tubular enclosure or demountable from the tubular enclosure. Preferably, the closure means is demountable from the tubular enclosure, and the closure means is positioned on the tubular enclosure after engaging a piece of separating gel into the tubular enclosure.
As disclosed in the parent, the closure means has an open end and an opposite end over which a semipermeable membrane, such as a dialysis membrane is secured. Other types of closure means membranes may also be used, such as an ion exchange membrane or other types of binding membranes, as long as the closure means membrane prevents the passage of nucleic acids and the like out of the tubular enclosure without hindering the electrical field of electrophoresis. The closure means membrane may be secured to the tubular enclosure or demountable closure means by using glue, a sleeve member, or other suitable means.
The inventor has improved upon the invention disclosed in the parent by providing a closure means having a passage means for reaching behind the closure means membrane and recovering the biomolecules accumulated against the closure means membrane during electroelution. "Behind the closure means membrane" and "behind the membrane" hereinafter refer to the face of the membrane where electroeluted biomolecules accumulate. The passage means is preferably an opening adjacent to the closure means opposite end. The passage means may be closed by the closure means membrane but is preferably closed by a separate membrane. If the closure means membrane is used to cover the passage means, a sleeve member is preferably used to secure the membrane over both the closure means and the passage means.
After electroelution is complete, the passage means may be opened by rupturing the membrane which covers the passage means. A pipette tip may be introduced through the passage means in order to reach behind the closure means membrane. The pipette tip is preferably positioned along side the closure means membrane and a thin film of buffer containing the electroeluted biomolecules is removed. The pipette tip used for reaching behind the closure means membrane preferably has a long capillary tip or needle.
The closure means may be provided with a constricted elution space between the tubular enclosure and the closure means membrane. The constricted elution space separates the gel pieces and the buffer in the tubular enclosure from the closure means membrane where electroeluted biomolecules are collected. The constricted elution space may be created by positioning a chamber or a porous septum between the tubular enclosure and the closure means membrane. The constricted elution space should have a capacity under 200 microliters, and preferably under 50 microliters.
The open ends of the tubular enclosure preferably have razor sharp edges to facilitate engaging pieces of the separating gel. If the tubular enclosure has demountable closure means, then both open ends of the tubular enclosure may be used for engaging pieces of gel and the two open ends may differ in the size of gel pieces the open ends engage. Preferably, the closure means is positioned over the end which engages the larger piece of gel.
The tubular enclosure and the closure means are preferably made of rigid and transparent materials which do not glow under ultra-violet (u.v.) light and preferably transmit u.v. light so that the DNA band may be observed by placing the tubular enclosure under u.v. light.
The method disclosed in the parent includes engaging the piece of separating gel containing the band of biomolecules intended for electroelution in the tubular enclosure by positioning the tubular enclosure over the separating gel directly above the band of biomolecules intended for electroelution. The tubular enclosure is then pushed through the soft separating gel thereby engaging the piece of separating gel containing the band of biomolecules intended for electroelution into the tubular enclosure. Preferably, the piece of separating gel is engaged into the tubular enclosure while the separating gel is submerged under the electrophoresis buffer. After engaging the separating gel piece, the piece is conveniently excised if the tubular enclosure is extracted from the separating gel by tilting it to a side.
After the piece of separating gel is engaged in the tubular enclosure the tubular enclosure is submerged in the electrophoresis tank. If the closure means is demountable from the tubular enclosure, after engaging the piece of separating gel in the tubular enclosure the closure means is capped on one or the tubular enclosure ends. Preferably no air bubbles are trapped in the tubular enclosure or the closure means. To avoid air bubble getting trapped in the tubular enclosure or the closure means, the tubular enclosure and the closure means should be filled with the electrophoresis buffer before being submerged in the electrophoresis tank. Preferably, the piece of separating gel is positioned close to the closure means or closure means membrane, allowing only a thin film of electrophoresis buffer between the gel piece and the closure means or closure means membrane. Preferably, the piece of separating gel in the tubular enclosure is pushed against the closure means or closure means membrane using a plunger type device (or plunger means), such as pipette tips or a bar. This forms the thin film of buffer between the gel piece and the closure means or closure means membrane.
As disclosed in the parent, after engaging the piece of separating gel in the tubular enclosure, the tubular enclosure is submerged in an electrophoresis tank such that the semipermeable closure means membrane faces the positive terminal. In fact, the tubular enclosure may be submerged in the same horizontal submerged electrophoresis device where the first or proceeding electrophoresis separation was performed. The tubular enclosure may be placed on top of the submerged separating gel.
When electrical current is applied to the electrophoresis tank the piece of separating gel engaged in the tubular enclosure experiences the electrophoresis electrical field. Biomolecules, such as protein and nucleic acids, in the piece of separating gel migrate toward the positive terminal and finally emerge out of the piece of gel into the buffer contained between the gel piece and the closure means membrane. Continuing the electrical field forces the biomolecules further toward the positive terminal which is blocked by the semipermeable dialysis membrane of the closure means. The semipermeable membrane prevents the migration of protein, nucleic acids and the like out of the tubular enclosure without hindering the electrophoresis electrical field, thereby concentrating protein and nucleic acids near the membrane. Because the nucleic acid molecules are visible under UV light, the migration can be followed under a UV lamp. The protein and nucleic acids accumulated near the closure means membrane may now be recovered with a pipette tip.
In the present invention, after electroelution is complete, the biomolecules accumulated against the closure means membrane may be recovered by rupturing the passage means with a sharp object, introducing a capillary pipette tip through the passage means to reach behind the closure means membrane. The pipette tip is preferably positioned along side the closure means membrane and the thin film of buffer (between the gel piece and the closure means membrane) containing the eluted biomolecules is removed. Any residual biomolecules may also be removed by applying a few drops of buffer or water on the gel piece and withdrawing the liquid with a pipette tip as described above.
The tubular enclosure and closure means of this invention accommodate electroelution in the same electrophoresis tank as where the first or preceding electrophoresis separation was performed. This eliminates the need for a separate electroelution device. The passage means of the present invention further assists the ease and accuracy of recovering the biomolecules.